GENETIC MUTATION

1. GENETIC MUTATION Asmarinah Department of Medical Biology Faculty of Medicine, University of Indonesia

2. DNA - Gene The gene store genetic information encoded in the sequence of nucleotide pairs in DNA Inheritance is based on genes that are transmitted from parents to offspring during reproduction with considerable accuracy

4. Mutation • Is the heritable changes in the genetic material. • The term mutation refers to • change in the genetic material, • the process by which the change occurs. Mutation provides the raw material for evolution. Without mutation, all of genes would exist in only one form and alleles would not exis. Organism would not be able to evolve and adapt to environment changes.

5. ▪ Mutations range in size from a single DNA building block (DNA base) to a large segment of a chromosome. ▪ An organism that exhibits a novel phenotype resulting from a mutation is called a mutant.

6. * Mutation may occur in any cell and at any stage in the development of a multicellular organism. - germinal mutation occur in germ-line cell and will be transmitted to progeny This type of mutation is present throughout a person’s life in virtually every cell in the body. - somatic mutation occur in somatic cell, occur in the DNA of individual cells at some time during a person’s life, andwill not be transmitted to the progeny.

7. Single-site or point mutation within individual gene • be classified: • - Substitution • In which one or more base-pairs are substituted with • other base pairs • Deletion • In which one or more base-pairs are lost • Insertion • In which one or more base-pairs are inserted into • sequence

8. Types of point mutation:

9. The mutation of a wild-type gene to a form that result in • a mutant phenotype forward mutation. • When a second mutation restores the original phenotype, • the process is called reverse mutation. • Reverse mutation may occur in two different ways, • by back mutation, a second mutation at the same site in the gene as the original mutation, restoring the wild type nucleotide sequence, • 2) by suppressor mutation, a second mutation at a different location in the genome, which compensates for the effects of the first mutation.

10. The deletion and insertion of one or two base-pairs within the coding sequence of a gene will alter the codon reading frame (i.e the series of base triplets that specify amino acid during translation) in mRNA.  frameshift mutations.

11. Mutation can alter the sequences of nucleotide pairs in genes, that cause changes in the amino acid sequences of the polypeptides  missense mutation. Mutation that alter the sequences of nucleotide pairs in genes, but can’t cause changes in the amino acid sequences of the polypeptides  silent mutation If the point mutation of base-pairs produce a stop codon (UAA, UAG and UGA) nonsense mutation

12. Many mutation have no effect on the phenotype of organism  neutral mutation. Most mutation with phenotypically recognizable effects result in decreased gene-product activity or no gene-product activity.

13. Mutation process and the expression of wild-type and mutant alles

14. Recessive mutant alleles often result in blocks in metabolic pathway • For example: 5 disorders are caused by autosomal recessive mutation with defect in phenylalanine-tyrosin metabolisme: • Phenylketonuria • Tyrosinosis • Tyrosinemia • Alkaptonuria • Albinism

16. Based on the cause of the mutation, there are: * Spontaneous mutation Occur infrequently, without a known cause. For eukaryotes, mutation rates range from about 10-7 to 10-9 per nucleotide pair per generation Mutation rate per gene varies from about 10-4 to 10-7 per generation * Induced mutation Resulting from exposure of organism to physical and chemical agents, called mutagens

17. Mutagenic agent (Mutagen) result in induced mutations. It is classified into: 1. Chemical - alkylating agent, that transfer alkyl group (CH3-, CH3CH2, and so forth) to the bases in DNA Example: Mustard gas [Di-(2-chloroethyl) sulfide] EMS (Ethyl methane sulfonate) - Basa analogs, that have similar structure to the normal base and are incorporated into DNA during replication Example: 5-Bromouracil (BU) 2-Aminopurin (2-AP)

18. Deaminating agent that deaminate the • amino groups in bases • Example: Nitrous acid (HNO2) • Hydroxylating agent, that hydroxylase amino • groups in the bases caused transition mutation. • Example: Hydroxylamine • Acridin dyes, that intercalate DNA molecule, so • doing, they increase the rigidity and alter the • conformation of the double helix • Example: Proflavin • Acridine orange

19. Some potent chemical mutagens

20. 2. Physical agents - Ionizing radiation High-energy rays collide with atom and cause the release of electrone, creating free radical or ion. It also induces gross changes in chromosome structure Examples: X-rays, gamma rays and cosmic rays - Nonionizing radiation Lower-energy that penetrate only the surface layer of cells and don’t cause ionizations. Example: UV light  cause pyrimidine hydrate pyrimidine dimer

21. 3. Transposable Genetic Elements (transposons) DNA element that can move from site in the genome to another site. The insersion of a transposons will often render the gene nonfunctional.

22. Three major factors for the occurring of mutation: • The accuracy of the DNA replication machinery • 2. The efficiency of the mechanisms that have evolved • for the repair of damaged DNA. • 3. The degree of the exposure to mutagenic agents • present in the environment.

23. The Mechanism of Mutation • Tautomerisation in DNA replication process • Tautomerisation is a process in which hydrogen atoms moves from one position to another position in a purine or pyrimidine . • Mutation resulting from tautomeric shifts cause: • - Transition mutation: replacement of purine with • other purine or of pyrimidine to other pyrimidine • - Transversion mutation: replacement of purine with • pyrimidine and vice versa

25. Tautomeric forms of the four common bases in DNA The shift of hydrogen atoms between the number 3 and 4 position of pyrimidine and between the number 1 and 6 position of the purine change their base-pairing potential

26. Mechanism by which tautomeric shift in the bases in DNA cause mutations

27. 2.Failure in DNA repair process DNA repair mechanisms are classified into: 1. Excision repair Consist of 3 steps: a. DNA glycosilase enzyme recognized and then excises the damage bases in DNA b. DNA polymerase fills nucleotide in the gap c. DNA ligase seals the break to complete the repair process

28. - Light-dependent repair (photoreactivation) DNA photolyase recognizes and binds to thymine dimers, and then uses light energy to cleave cross-link

29. Inherited human disease with DNA mutation (examples) • “Sickle cell anemia” : • Pauling et al., (1949) discovered: substitution of nucleotide A to T that substitute amino acid glutamic acid in the normaly hemoglobin (Hb A) to valine (defective hemoglobin (Hb S). •  introduced “molecular disease” • term • set the foundation of molecular diagnosis

30. - “Tay-Sachs disease”: mutation in the HEXA gen which encodes the enzyme hexosamidase A to convert ganglioside GM2 into GM3 in nerve cells. In the absence of the enzyme, gangliosideGM2accumulates and blocks neuron activity.

31. Inherited human disease with defects in DNA repair • Xeroderma Pigmentosum • extremely sensitive to sunlight. It caused by defect in • the repair of UV-induced damaged to DNA • Cockayne syndrome • Defect in nucleotide excision repair

32. Mutation is one of sources for polymorphism • Polymorphism • “many form” : a set of two or more alternative of normal phenotypes Genetic polymorphism: The occurrence of two or more allele in a locus in which its frequency is more than 1% in population. Example: ABO and MN blood group If the alleles with frequencies of less than 1% are called rare variants . Example: O Bombay blood group

33. ▪ Polymorphisms are responsible for many of the normal differences between people such as eye color, hair color, and blood type. ▪ Although many polymorphisms have no negative effects on a person’s health, some of these variations may influence the risk of developing certain disorders.

34. Forms of genetic polymophism • Single Nucleotide Polymorphism (SNP), a polymorphism in DNA sequence consisting of variation in a single base. • Example: Missence mutation that caused by a nucleotide substitution

35. 2. Variable number of tandem repeats (VNTR), a type of DNA polymophism created by a tandem arrangment of multiple copies of short DNA sequences.

36. 3. Microsatellites (Short Tandem Repeat Polymorphism = STRP) A polymorphic locus consisting of a variable number of tandemly repeated bi-, tri-, or tetranucleotide units such as (TG)n, (CAA)n, or (GATA)n.  Different numbers of units constitute the different alleles

37. Gene polymorphism • Occur in 1 in 1000 DNA bp in human genome  over 3 million SNPs in the entire human genome • Is reflected in the diversity of the gene products, such as structural protein, enzymes, channel proteins, receptors, etc.

38. Use of polymorphism • Medical forensic  individual characteristic • Disease  suceptibility, influence drug response • Genetic relationship (within & between population) •  molecular evolution

39. Genetic Mutation/Polymorphism can be detected by following techniques: * RFLP (Restriction Fragment Length Polymorphism) * PCR (Polymerase Chains Reaction) – RFLP * PCR – DNA Sequencing * Southern Blot

40. 1300 1100 Sickle-cell disease

41. 174 Mutation in exon 6 of VDAC3 gene in sperm with low motility Sequence of PCR product from sperm with normal motility Posisition 174 : AAG (Lysine) Sequence of PCR product from sperm withlowmotility Posisition 174 : GAG (glutamic acid)

43. References: • Alberts et al., 2008. Molecular Biology of the Cell. 5th ed. • Karp. 2005. Cell and Molecular Biology. 4th ed. • Nussbaum et al. 2001. Thompson & Thompson Genetic Medicine. 6th ed. • Snustad & Simmons. 2003. Principles of Genetics . 3rd ed.